Treatments that activate immune responses against tumor cells have revolutionized cancer therapeutics. These treatments are the culmination of decades of studies into how the immune system recognizes and eliminates cancer, but more research is necessary to find additional therapeutic targets and improve current ones. Natural Killer (NK) cells are one type of immune cell with potent anti-tumor activities. NK cells delineate cancer cells from normal tissue using germline-encoded cell surface receptors that recognize molecules on transformed cells but not healthy cells. NK cells eliminate tumors by secreting cytotoxic granules and powerful anti-tumor cytokines. Because many therapies to boost NK responses are in clinical development, it is crucial to thoroughly explore the mechanisms that regulate NK activity against tumors. A recent study in our laboratory provided evidence that tumor-infiltrating myeloid cells persistently stimulate NK cells through the NK immunoreceptor NKG2D. This persistent stimulation causes NK cells to be less active and unable to kill cancer cells, allowing the tumor to evade NK recognition. This desensitization is driven by the NKG2D ligand (NKG2D-L) RAE-1? expressed on tumor-infiltrating macrophages and monocytes. Preliminary data suggest that myeloid RAE-1? expression is caused by the cytokine MCSF, produced by tumor cells. Thus, tumors may facilitate their own growth using a multicellular axis to circumvent NK cell-mediated immunosurveillance. This application seeks to further explore the molecular underpinnings of these findings, and to test whether interrupting NKG2D/RAE-1? interactions between NK cells and myeloid cells can relieve this desensitization and promote tumor rejection. In Aim 1, blocking antibodies against MCSF, lentiviral overexpression of MCSF, and Cas9-mediated knockout of the MCSF gene in tumor cells will be used to determine whether tumor-derived MCSF promotes RAE-1? expression on tumor-infiltrating myeloid cells in transplanted and autochthonous tumor models. The signaling pathways responsible for RAE-1? induction by MCSF will be explored by examining whether PI3K, downstream of MCSF stimulation, regulates RAE-1? in macrophages. Overexpression and knock-down strategies will be used to modulate PI3K signals to examine the contribution of this pathway to RAE-1? induction. Finally, I will determine whether human MCSF can stimulate NKG2D-L induction on peripheral blood monocytes, and examine human NKG2D-L expression on myeloid cells infiltrating tumors in humanized mice. In Aim 2, blocking antibodies against MCSF and RAE-1?, genetic ablation of RAE-1?, and macrophage depletion will be used to determine whether interrupting the MCSF/RAE-1?/NKG2D desensitization axis, alone or in combination with other therapies that activate NK cells, can promote rejection of tumors expressing ligands for NKG2D and other NK receptors. We hypothesize that MCSF- and RAE-1?-dependent NK desensitization is a common feature of tumors and a potentially worthwhile target for cancer therapy.